Individuals exposed to maternal diabetes in utero are more likely to develop metabolic and cardiovascular diseases later in life. This may be partially attributable to epigenetic regulation of gene expression. To examine whether differential DNA methylation, a major source of epigenetic regulation, can be observed in offspring of mothers with type 2 diabetes (OMD) during the pregnancy as compared to offspring of mothers with no type 2 diabetes(OMND) during the pregnancy, we performed an epigenome-wide association study. A total of 423,311 cytosine-phosphate-guanine (CpG) sites were analyzed in 388 Native Americans, of which 187 were OMD and 201 were OMND. Forty-eight differentially methylated CpG sites (with empirical false discovery rate < 0.05), mapping to 29 genes and 10 intergenic regions, were identified. The gene with the strongest evidence was LHX3, where 6 CpG sites were hyper-methylated in OMD (P 1.1 10-5). Similarly, a CpG near PRDM16 was hyper-methylated in OMD (1.1% higher, P = 5.6 10-7), where hyper-methylation also predicted future diabetes risk (hazard ratio = 2.12, P = 9.7 10-5). Hyper-methylation near AK3L1 and hypo-methylation at PCDHGA4 and STC1 associated with exposure to diabetes in utero and decreased insulin secretory function among offspring with normal glucose tolerance. Analysis of all 29 genes in aggregate did not shown enrichment for any biologic pathway; however, literature searches provided evidences that several of these genes have a role in embryonic development and/or beta cell function. We conclude that intrauterine exposure to diabetes can affect methylation at multiple genomic sites. Methylation status at some of these sites can impair insulin secretion, increase body weight and increase risk of type 2 diabetes.